Because of their capacity to provide long-term in vivo reconstitution of the blood system, hematopoietic stem cells are an attractive target for somatic gene therapy. Retroviral gene transfer into cord blood or marrow cells has been used to correct genetic defects most successfully in patients with potentially fatal human severe combined immunodeficiency disease (SCID). In a total of 13 children with X-linked SCID worldwide, T cell and immune function could be restored to normal by transplantation of genetically corrected bone marrow cells expressing the human IL-2-receptor gamma chain (IL-2Rgamma) cDNA. Retroviral gene therapy of another form of SCID (ADA-SCID) has now also obtained significant levels of T cell correction. The emergence of two cases of a T-cell Acute Lymphocytic Leukemia like disease (T-ALL) that is most likely secondary to the genetic modification in the two youngest patients of the X-SCID gene therapy trial conducted by Fischer and colleagues has prompted new critical questions into the basis and generalizability of this phenomena in genetically modified hematopoietic cells. We have developed a new linear amplification mediated (LAM) PCR method that has allowed us to identify single retroviral insertion sites from freshly transduced cells in vitro, and in vivo, the proliferative expansion of single stem cell clones. This approach allowed us to discover retroviral insertion-activation of the LMO2 gene, a T-ALL associated oncogene, in the two patients suffering from the leukemic syndrome in the Fischer trial. Preliminary data obtained by the analysis of additional insertion sites from these and other patients suggests that a broad assessment of retroviral insertions before and after in vivo clone selection can be accomplished and that this will allow us to gain substantial additional insight into the identity, significance, and impact of retroviral insertions in gene corrected progenitor cells. Using material from all patients in these two clinical gene transfer trials, and insertion within the LMO2 locus as a model system, it is the overall aim of this proposal to determine the frequency of retrovirus insertion into the vicinity of the LMO2 locus in human repopulating cell clones and to track their fate in vivo (Aim 1), to understand the frequency of insertions into other critical cellular genes and gene structures (Aim 2), and to map the clonal composition of the inventory of genetically corrected hematopoietic cells in typical patients of the X-SCID gene therapy trials (Aim 3) in order to develop safe and efficient dosage and side effect prevention strategies for future gene therapy trials. Results from these studies of insertion sites in the human genome, and the role of LMO2 overexpression on the evolution of a lymphoproliferative phenotype will have great significance for the field.